Traditionally, cell separation technologies include active and passive cell separation technologies, wherein the active cell separation technology means that the target cells are filtrated or separated by different approaches, such as dielectrophoresis, optical tweezers, and magnetic force. Furthermore, most of the passive cell separation technologies mean that the target cells are filtrated or separated by sieve elements, and the size of meshes on sieves can prevent target cells from passing through the sieves.
For example, referring now to Taiwan Pat. No. I308131, a bio-particle capturing apparatus having a 3D structure and a manufacturing method thereof are disclosed, wherein the capturing apparatus is a trap to capture bio-particles to the predetermined wells by dielectrophoresis (DEP) force. The characteristic of the capturing apparatus is to apply a 3D-structural concept to a dielectrophoresis biochip which is mainly used to trap and immobilize bio-particles, such as cells, functional latex beads, nano-particles or gene segments.
As described in the Taiwan Pat. No. I308131, the bio-particles capturing apparatus having the 3D structure comprises an upper layer, microfluidic channels and a lower layer, wherein the upper layer is formed with an upper electrode, an inlet and an outlet; and the lower layer is formed with a lower electrode. A sample fluid can flow from the inlet into the capturing apparatus, flow through the microfluidic channels, and then flow out of the outlet of the capturing apparatus. Furthermore, the outlet is formed with matrix-type wells. The major characteristic of the capturing apparatus is that the directions of electric fields generated by the electrodes of the upper and lower layers are vertical to the flow direction of the sample fluid of the microfluidic channels, so as to form uneven longitudinal electric fields. Thus, the bio-particles in the microfluidic channels can be rapidly captured into the predetermined wells of the low layer.
However, the bio-particle capturing apparatus having the 3D structure is a trapping device which uses the DEP force generated by the electrode to trap the bio-particles into the predetermined wells. In actual use, a buffer with lower conductivity (˜570 μS/cm) must be applied to the capturing apparatus for trapping or separating the bio-particles. For example, the conductivity of human blood is about 0.1˜2 S/cm, and thus does not meet the condition of buffer with the lower conductivity for the trapping or separating the bio-particles by using DEP force. Consequently, before the cell separation, it needs to firstly separate the cells of blood by the method of density gradient separation. After centrifugation, the collected cells are added into the lower conductivity buffer, so that the more powerful DEP force can be generated to trap the target cells.
As described above, except for actively trapping the target cells of blood samples by the DEP force, magnetic particles also can be immobilized on cell membrane, or the magnetic particles can be introduced into the cells by uptake events, so target cells can be separated by manipulated magnetic force. However, most of the biological features of tumor cells are almost the same as that of health cells. Therefore, it is necessary to screen a high specific protein for particular membrane protein of particular cells at first, conjugate the magnetic particles to the specific protein, and then to apply the property of the specific protein able to identify the particular membrane protein of the particular cells for immobilizing the magnetic particles onto the cell surface of particular tumor cells as to-be-screened targets. Moreover, if using the method of inducing the particular cells to actively take into the magnetic particles as to-be-screened targets, a particular structural molecule must be modified on the surface of the magnetic particles, so that the specific cells are promoted to swallow them. However, no matter for modifying the surface of the magnetic particles or conjugating the magnetic particles to the specific protein, the particular magnetic particles must be added during screening different target cells, so that the cost of detection will be increased.
Besides, the easier and more convenient method of separating the bio-particles is to use a sieve apparatus to the passive separation of bio-particles. That is, the size of the meshes defined on the sieve apparatus can be used to screen bio-particles with different diameters in a sample fluid. It is unnecessary to execute any pre-treating steps before passively screening the sample fluid, the time cost of screening or separating the target bio-particles can be relatively decreased, and the manufacture cost of the sieves can be lowered by mass production of the sieves.
Recently, 2D or 3D sieve apparatus made by micro-electro-mechanical systems (MEMS) technology are widely applied to separate cells, wherein the meshes of the sieve are used to trap white blood cells of blood; and red blood cells, platelets and serum of blood can pass through the meshes of the sieves, so as to carry out the purpose of screening and separating the white blood cells. However, in fact, for a sieve apparatus made of silicon nitrite or parylene C and processed by MEMS technology, during forming the 3D sieve structure, a sacrificial layer must be sandwiched between an upper sieve and a lower sieve. The sacrificial layer is selectively etched after forming upper meshes on the upper sieve. Therefore, the process of the 3D sieve apparatus must consider the ratio of the etching rate between materials of the upper sieve, the lower sieve and the sacrificed layer. Moreover, it also needs to consider the three materials of the 3D sieve apparatus with the problems of residual stress generated during the manufacture process and to consider relative parameters thereof, so that it is significantly difficult in the whole manufacturing process of the 3D sieve apparatus.
As a result, the traditional sieve apparatus manufactured, by the MEMS technology is not only complicated and difficult to execute MEMS processes, but also the stability of the manufacture yield thereof is still not good. Therefore, it is difficult to lower the manufacture cost of industrial mass production. Therefore, it is necessary to provide a miniature sieve apparatus and a manufacturing method thereof to solve the problems existing in the conventional sieve apparatus, as described above.